Design of Heme Protein-Based Blood Substitutes

描述（由申请人提供）：我们的长期目标是开发重组人血红蛋白（rHb）作为生产所有基于球蛋白的O2输送药物的起始材料。我们已经确定了基于rHb的血液替代品的关键要求：（a）中等的O2亲和力和大的O2解离速率常数，以在毛细血管中有效转运;（B）显著降低NO清除速率，以防止高血压副作用;（c）对自身和化学诱导的氧化的抗性，以抑制氧化应激;和（d）低的血红素解离速率，以增加保质期。我们已经使用这些属性的机制来设计第二代细胞外rHb为基础的血液替代品，更有效的O2运输和很少或没有高血压的副作用，在临床前动物研究。然而，显然需要工程化第三代非血管活性rHb，其具有增加的变性抗性、增强的表达产率和降低的生产成本。我们提出了解决这些问题的方法：（1）优化人rHb与O2的结合和NO的清除，同时不影响球蛋白的稳定性和对降解反应的抗性;（2）提高重组血红蛋白在E.通过（a）合理的和比较性的诱变以增加脱辅基蛋白对解折叠的抗性和（B）α和β rHb链与新发现的红细胞伴侣α血红蛋白稳定蛋白（AHSP）的共表达，在大肠杆菌中表达;和（3）通过rHb与血红素利用基因（hug）的共表达，促进外源血红素快速掺入新合成的球蛋白中来自类志贺邻单胞菌和相关病原体。公共卫生：我们正在开发重组人血红蛋白，以取代捐献的血液，作为所有正在开发用于治疗严重失血的蛋白质氧载体的源材料，并认为这一努力应成为国家公共卫生的优先事项。一种有效的基于血红蛋白的血液替代品将缓解全血的长期短缺，在军事和民用灾难后提供紧急治疗，并允许在缺乏安全血库系统的发展中国家使用输血。

DESCRIPTION (provided by applicant): Our long-range goal is to develop recombinant human hemoglobin (rHb) as the starting material for manufacturing all globin-based O2 delivery Pharmaceuticals. We have established the key requirements for a rHb-based blood substitute: (a) moderate O2 affinity and large O2 dissociation rate constants for efficient transport in capillaries; (b) significantly reduced rates of NO scavenging to prevent hypertensive side effects; (c) resistance to auto- and chemically-induced oxidation to inhibit oxidative stress; and (d) low rates of heme dissociation to increase shelf-life. We have used the mechanisms governing these properties to design second-generation extracellular rHb-based blood substitutes with more efficient O2 transport and little or no hypertensive side effect in pre-clinical animal studies. However, there is a clear need to engineer third-generation, non-vasoactive rHbs with increased resistance to denaturation, enhanced expression yields, and reduced production costs. We propose to solve these problems by: (1) optimizing O2 binding to and NO scavenging by human rHb without compromising globin stability and resistance to degradative reactions; (2) improving the stability and enhancing production of recombinant hemoglobin in E. coli by (a) rational and comparative mutagenesis to increase the resistance of the apoprotein to unfolding and (b) co- expression of the alpha and beta rHb chains with the newly discovered erythroid chaperone, alpha hemoglobin stabilizing protein (AHSP); and (3) facilitating rapid incorporation of exogenously added heme into newly synthesized globins by co-expression of rHb with the heme utilization genes (hug) from Plesiomonas shigelloides and related pathogens. PUBLIC HEALTH: We are developing recombinant human hemoglobin to replace donated blood as the source material for all protein-based oxygen carriers that are being developed for the treatment of severe blood loss and believe that this effort should be a national public health priority. An effective hemoglobin- based blood substitute will alleviate chronic shortages of whole blood, provide emergency treatment following military and civilian disasters, and allow the use of transfusions in developing countries lacking a safe blood banking system.